Composite containing collagen and elastin as a dermal expander and tissue filler

ABSTRACT

An injectable composition having dermal filling and tissue expanding activity comprises: (1) a quantity of elastin sufficient to bring about dermal filling and tissue expansion when injected into a subject in need of dermal filling and tissue expansion; and (2) a pharmaceutically acceptable carrier. The composition can further comprise collagen; in other alternatives, the composition can further comprise hyaluronic acid; and one or more of the elastin, the collagen, and the hyaluronic acid, if present can be cross-linked, either intramolecularly or intermolecularly. The elastin, however, is the primary filler, even if collagen or hyaluronic acid are included in the composition. Proanthocyanidin can act as a cross-linker and as a protector of elastin against UV degradation. Proanthocyanidin combined with a collagen or gelatin-hyaluronic acid matrix can inhibit matrix degradation and contraction with fibroblasts. Additionally, the present invention encompasses a method for augmenting the volume area of dermal tissue at sites where wrinkles and other tissue imperfections have caused visible changes associated with loss or rearrangement of the subcutaneous collagen network comprising the step of: providing an effective amount of a composition, which can be injected locally, and which consists of a mixture of macromolecules which are native constituents of the skin, the mixture comprising collagen, elastin and hyaluronic acid, which are held together by covalent, ionic or hydrogen bonds, and which include natural materials which have been demonstrated to increase the natural synthesis of these molecules by cells which reside in the dermis.

CROSS-REFERENCES

This application is a divisional application of U.S. patent applicationSer. No. 15/012,257 by Han et al., entitled “Composite ContainingCollagen, Elastin, and Hyaluronic Acid as a Dermal Expander and TissueFiller” and filed on Feb. 1, 2015, which was a divisional application ofU.S. patent application Ser. No. 13/127,275 by Han et al., entitled“Composite Containing Collagen, Elastin, and Hyaluronic Acid as a DermalExpander and Tissue Filler” and filed on Sep. 23, 2011, now U.S. Pat.No. 9,248,165, issued on Feb. 2, 2016, which was a United Statesnational stage application under 35 U.S.C. § 371 of PCT ApplicationSerial No. PCT/US2009/063132 by Han et al., entitled “CompositeContaining Collagen, Elastin, and Hyaluronic Acid as a Dermal Expanderand Tissue Filler” and filed on Nov. 3, 2009, which, in turn, claimedthe benefit of U.S. Provisional Application Ser. No. 61/111,637 by Hanet al., entitled “Composite Containing Collagen, Elastin, and HyaluronicAcid as a Dermal Expander and Tissue Filler” and filed on Nov. 5, 2008.The contents of these applications are hereby incorporated herein intheir entirety by this reference.

FIELD OF THE INVENTION

This invention is directed to a composition for injection that acts as adermal expander and tissue filler. The composition is a composite thatcontains collagen, elastin, and hyaluronic acid; the collagen, elastin,and hyaluronic acid can be covalently cross-linked; in particular, theelastin is cross-linked or otherwise stabilized by interaction with aproanthocyanidin.

BACKGROUND OF THE INVENTION

As the skin ages, many people experience dissatisfaction in theirappearance due to the development of lines and wrinkles in the skin,especially the face. Although the development of these lines andwrinkles is generally seen as a part of the natural aging process, formany individuals, the appearance of these lines and wrinkles is verytraumatic and seriously affects their quality of life and theirrelationships with others. In addition, for a large number of people,the appearance of aging can affect their economic prospects. This isespecially true for those seeking employment as actors or actresses orin other positions in the entertainment industry, but there is anincreasing concern among an aging population that an aged appearance canaffect their job prospects, even for jobs for which appearance isessentially unrelated to job performance.

Therefore, as a result of these concerns, there is a substantial needfor products that can reverse some of the effects of the aging processand can act as dermal expanders and tissue fillers. Accordingly, anumber of compositions are in use as dermal expanders and tissuefillers. Some of these are collagen-based. One alternative usesatelopeptide collagen alone, such as Zyderm (from bovine collagen), ormicronized human collagen from cadavers. Another alternative usescross-linked collagen, such as Zyplast, which uses bovine collagencross-linked by glutaraldehyde, or Evolence, which uses porcine collagencross-linked by ribose. Other compositions in use as dermal expandersand tissue fillers are hyaluronic acid-based, either native orcross-linked. These dermal expanders and tissue fillers have severalproblems. They have a great tendency to be resorbed, require repeatedinjections, and there is a risk of toxicity from the cross-linkingagents used.

Another approach uses composites. These include, for example, CaHAmicrospheres suspended in a gel carrier (Radiesse), PMMA beads suspendedin gelatin (ArteFill), PLA beads, TCP powder, or dextran beads inhyaluronic acid (HA). These composites also have several problems. Forexample, the beads used are non-biological and tend to migrate, whichcauses distortion of the appearance of the skin.

There are additional uses for dermal expanders and tissue fillers thatrequire dermal expanders or skin fillers with improved properties. Theseare in breast augmentation, treatment for urinary incontinence, ortreatment for esophageal reflux. Such dermal expanders and fillers canbe employed in other surgical reconstructive processes. Such dermalexpanders and fillers can also be employed in dental implants, wherethey can aid in bone regeneration.

Accordingly, there is a need for an improved dermal expander and tissuefiller that is effective, safe, and whose effects are lasting. There isalso a need for an improved dermal expander and tissue filler that canbe employed in non-cosmetic applications such as breast augmentation,treatment for urinary incontinence, or treatment for esophageal reflux.

SUMMARY OF THE INVENTION

The present invention describes improved dermal expanders and tissuefillers that meet the needs described above.

Injectable implant compositions for soft tissue augmentation comprisecollagen (obtained from skin, tendon or bone) and elastin (obtained fromarteries or tendons and ligaments). The elastin is the primary filler.The compositions are suspended in a physiological saline solution forintradermal injection or other soft tissue augmentation. The compositein question is retained at the site of injection for prolonged periodsof time for the purpose of eliminating wrinkles or other visible surfaceimperfections in the skin. Other ingredients can be included, such as,but not limited to, hyaluronic acid.

Since the 1980's injectable collagen, usually from bovine sources, butmost recently also of human origin, has been used as at tissue filler toeliminate wrinkles and other facial imperfections, usually associatedwith the aging process. In recent years other natural, synthetic andinert fillers have been used for the same purpose. Presently the use ofthese alternate materials exceeds that of bovine collagen, which isbeing replaced by materials which are more lasting and have a lessertendency to be reactive and induce allergic reactions. Hyaluronic acidand its derivatives, principally cross-linked hyaluronic acid, aregaining more and more acceptance and are becoming the most widely usedtissue fillers. Among these, non-animal derived hyaluronic acid isreceiving the greatest acceptance because of the very small incidence ofallergic reactions and the almost immediate ability to correct facialwrinkles or folds. Most of these acceptable currently used fillers willretain satisfactory beneficial results for periods longer than 6 monthsand up to 1 year, at which time application has to be repeated.

Elastin is an important skin component which contributes to the naturalproperties of skin, and it is one of the dermal components which morereadily becomes depleted during the process of aging. Because of thenature of its chemical composition, and the uniqueness of itscross-linking network, which contributes to make it one of the mostinsoluble proteins in the body, it is very resistant to chemical andenzymatic degradation. Nevertheless UV light has the tendency to destroythe complex cross-links, opening the ring structure of pyridine groupswhich stabilize the protein structure. Sunlight exposure is known toinduce a phenomenon, described as solar elastosis, which causesdegradation and loss of structure of skin elastin and alters theproperties of collagen. The phenomenon is very well described and hasbeen studied extensively in the weathered skin of individuals who areconstantly exposed to sunlight and consequent to sun induced skindamage. In some alternatives, a proanthocyanidin is added, which,because of its antioxidant properties, is expected to further protectthe injected elastin from UV degradation. When elastin is used, it ishighly preferred to cross-link the elastin with a proanthocyanidin.Preferably, a proanthocyanidin is used at a concentration of from about0.1% to about 1.0%. The pH is preferably from about 7.0 to about 8.5.The interaction of the proanthocyanidin can be described as stabilizing,cross-linking, or coating. The use of a proanthocyanidin to cross-link,stabilize, or coat elastin in a microparticle suspension is highlypreferred in order to prevent its biodegradation. If not cross-linked,stabilized, or coated, elastin degrades rapidly, as determined by an invitro enzyme degradation test.

In the present invention we have combined collagen and elastin in theformulation. The natural resistance of elastin to degradation, coupledwith its space filling ability when combined with collagen satisfies thecriteria of ideal filler. Elastin is the primary filler. It contains allthe natural materials present in the dermis to achieve the ultimate goalof retaining a smooth outer appearance of the skin by eliminating theempty spaces and reversing the contractile process that is responsiblefor the superficial uneven appearance of skin. In some alternatives, aglycosaminoglycan such as hyaluronic acid can be included as well; insuch alternatives, the collagen and the hyaluronic acid can form acollagen-hyaluronan complex.

The present invention contains a mixture of collagen, elastin, and theglycosaminoglycan or any of their combinations.

Collagen can be in its native form, in a denatured form (gelatin) orcross-linked endogenously or exogenously.

Collagen can be obtained from a variety of animal and human tissues,such as skin, tendon, ligament, pericardium, dural membrane, smallintestine mucosa, and bone, as well as other tissues.

Elastin can be obtained from animal and human tendon, such as bovineligamentum nuchae, aorta roots, blood vessels, pericardium, as well asother tissues.

Accordingly, one aspect of the invention is an injectable compositionhaving dermal filling and tissue expanding activity comprising:

(1) a quantity of elastin sufficient to bring about dermal filling andtissue expansion when injected into a subject in need of dermal fillingand tissue expansion; and

(2) a pharmaceutically acceptable carrier.

Typically, the elastin is selected from the group consisting ofnon-human mammalian elastin and human elastin. If the elastin isnon-human mammalian elastin, it is typically porcine elastin.

The pharmaceutically acceptable carrier can comprise gelatin. It canfurther comprise phosphate buffered saline or other excipients; otherexcipients such as glucose or maltose have been described and are knownin the art. In another alternative, the pharmaceutically acceptablecarrier can comprise carboxymethylcellulose. Other acceptable carriersare known in the art.

In one preferred alternative, the composition further comprises arapid-acting local anesthetic. The rapid-acting local anesthetic can beselected from the group consisting of lidocaine, benzocaine, tetracaine,bupivacaine, cocaine, etidocaine, flecainide, mepivacaine, pramoxine,prilocaine, procaine, chloroprocaine, oxyprocaine, proparacaine,ropivacaine, dyclonine, dibucaine, propoxycaine, chloroxylenol,cinchocaine, dexivacaine, diamocaine, hexylcaine, levobupivacaine,pyrrocaine, risocaine, rodocaine, and pharmaceutically acceptablederivatives and bioisosteres thereof. Typically, the rapid-acting localanesthetic is lidocaine.

The elastin can be cross-linked with a cross-linking agent. Thecross-linking agent can be selected from the group consisting of aproanthocyanidin, a bifunctional epoxide, a carbodiimide,glutaraldehyde, and periodate. If the cross-linking agent is aproanthocyanidin, it is typically selected from the group consisting ofproanthocyanidin, procyanidin (2H-1-benzopyran-3,4,5,7-tetrol,2-(3,4-dihydroxyphenyl)-2-(2-(3,4-dihydroxyphenyl)-3,4-dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl)oxy)-3,4-dihydro),procyanidin B, procyanidin B2, rhatannin, procyanidol oligomer,procyanidin C, procyanidin B3, procyanidin B1, selligueain A(8,14-methano-2H,14H-1-benzopyrano(7,8-d)(1,3)benzodioxocin-3,5,11,13,15-pentol,4-(3,4-dihydro-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-2H-1-benzopyran-8-yl)-3,4-dihydro-2,8-bis(4-hydroxyphenyl)-,(2R-(2α,3α,4β(2R*,3S*),8β,14β,15R*)),geranin A, geranin D, procyanidin B5, procyanidin B5-3′-O-gallate,vitisinol, amurensisin, terminalin, geranin B, 6,8-dihydroxyafzelin,afzelin-3″-O-gallate, geranin C, afzelin, flavangenol, carallidin,mahuannin A, proanthocyanidin A1, proanthocyanidin A2, procyanidin D,and analogues, derivatives, and bioisosteres of these compounds.Preferably, a proanthocyanidin is used at a concentration of from about0.1% to about 1.0%. The pH is preferably from about 7.0 to about 8.5.The interaction of the proanthocyanidin can be described as stabilizing,cross-linking, or coating. In most embodiments of the invention, it ishighly preferred to use a proanthocyanidin as a cross-linking agent.However, other cross-linking agents can be used. If the cross-linkingagent is a bifunctional epoxide, it is typically 1,4-butanedioldiglycidyl ether. If the cross-linking agent is a carbodiimide, thecarbodiimide is typically selected from the group consisting of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide (EAC);1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl-carbodiimidemetho-p-toluenesulfonate (CMC), andN-benzyl-N′-3-dimethylaminopropyl-carbodiimide hydrochloride (BDC).Preferably, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

In one alternative, the elastin is cross-linked with aglycosaminoglycan. The glycosaminoglycan can be selected from the groupconsisting of hyaluronic acid, chondroitin sulfate, pentosanpolysulfate, dermatan sulfates, heparin, heparan sulfates, and keratansulfates. Typically, the glycosaminoglycan is hyaluronic acid. Suitablecross-linking agents are as described above. When the glycosaminoglycanis hyaluronic acid, the hyaluronic acid can be obtained from roostercombs or can be synthesized by bacteria.

In another alternative, the composition further comprises collagen. Thecollagen can be native or denatured collagen obtained from skin, tendon,ligament, pericardium, dural membrane, small intestine mucosa or bone.The collagen can be used in soluble form, fibrillar form, a form of aninsoluble slurry, or hydrogel form. The collagen can be human Type Icollagen. The human Type I collagen can be obtained from demineralizedbone matrix (DBM). Alternatively, the collagen can be collagen fromporcine tendon. The collagen can be cross-linked intramolecularly.Suitable cross-linking agents are described above. Alternatively, thecollagen can be cross-linked to the elastin, or the collagen can becross-linked to a glycosaminoglycan such as, but not limited to,hyaluronic acid, which can also be included in the composition in thatalternative.

The elastin can be obtained from human or animal ligaments or arteries.Typically, the elastin is purified from non-elastineous proteins andproteoglycans. Typically, the elastin is cryofractured into a fineparticle. Preferably, the size of the fine particle is from about 50 μmto about 210 μm. Typically, the elastin concentration is from about 20mg/mL to about 300 mg/mL; preferably, the elastin concentration is fromabout 20 mg/mL to about 100 mg/mL.

In one alternative of this aspect of the invention, the compositionfurther comprises collagen and a glycosaminoglycan such as, but notlimited to, hyaluronic acid, and wherein one or more of the elastin, thecollagen, and the glycosaminoglycan are cross-linked, eitherintramolecularly or intermolecularly. Suitable cross-linking agents aredescribed above.

Another aspect of the invention is a method of dermal filling and tissueexpansion comprising the step of injecting a quantity of this embodimentof a composition according to the present invention as described abovesufficient to induce dermal filling and tissue expansion to a patient inneed thereof. In this method, the dosage, the frequency of the dosage,and the route of injection can be chosen by a skilled clinician takinginto account factors known in the art and described in detail furtherbelow.

Another aspect of the present invention is an injectable compositionhaving dermal filling and tissue expanding activity comprising:

(1) an insoluble collagen suspension in a quantity sufficient to bringabout dermal filling and tissue expansion when injected into a subjectin need of dermal filling and tissue expansion; and

(2) a pharmaceutically acceptable carrier.

Typically, in this composition, the concentration of collagen is fromabout 20 mg/mL to about 100 mg/mL. Typically, in this composition, thecollagen is cross-linked with a cross-linking agent, as described above.

Typically, in this composition, the pharmaceutically acceptable carrieris gelatin.

In this aspect of the invention, the composition can further compriseelastin. In one preferred alternative when the composition compriseselastin, the composition comprises 75 mg of elastin particles and 25 mgof insoluble collagen per 1.0 mL of pharmaceutically acceptable carrier.In this alternative, the pharmaceutically acceptable carrier can begelatin.

In this aspect of the invention, the collagen can be partially denaturedinto gelatin to aid in maintaining the components in suspension and infacilitating the injection of the mixture through a narrow gauge needle.

Another aspect of the invention is a method of dermal filling and tissueexpansion comprising the step of injecting a quantity of this embodimentof a composition according to the present invention as described abovesufficient to induce dermal filling and tissue expansion to a patient inneed thereof. In this method, the dosage, the frequency of the dosage,and the route of injection can be chosen by a skilled clinician takinginto account the factors described further in detail below.

Yet another aspect of the present invention is a method for augmentingthe volume area of dermal tissue at sites where wrinkles and othertissue imperfections have caused visible changes associated with loss orrearrangement of the subcutaneous collagen network comprising the stepof: providing an effective amount of a composition, which can beinjected locally, and which consists of a mixture of macromoleculeswhich are native constituents of the skin, the mixture comprisingcollagen, elastin and, optionally, a glycosaminoglycan, such as, but notlimited to, hyaluronic acid, which are held together by covalent, ionicor hydrogen bonds, and which include natural materials which have beendemonstrated to increase the natural synthesis of these molecules bycells which reside in the dermis. In this method, the collagen can benative or denatured collagen obtained from skin, tendon, ligament,pericardium, dural membrane, small intestine mucosa or bone. Thecollagen can be used in soluble form, fibrillar form, a form of aninsoluble slurry, or hydrogel form. The collagen can be cross-linkedintramolecularly. Alternatively, the collagen can be cross-linked to theelastin or to the glycosaminoglycan, such as, but not limited to,hyaluronic acid. The collagen can be insoluble collagen; when thecollagen is insoluble collagen, the concentration of insoluble collagenis typically from about 20 mg/mL to about 100 mg/mL.

The elastin used can be obtained from human or animal ligaments orarteries. The elastin is typically purified from non-elastineousproteins and proteoglycans. The elastin is typically cryofractured intoa fine particle. Typically, the size of the fine particle is from about50 μm to about 210 μm.

In this method, the injectable composition used typically comprisesgelatin.

In this method, typically, the elastin concentration is from about 20mg/mL to about 100 mg/mL.

In this method, when the glycosaminoglycan is hyaluronic acid, thehyaluronic acid used can be obtained from rooster combs or issynthesized by bacteria.

In this method, at least one of the collagen, the elastin, and, ifpresent, the glycosaminoglycan, such as, but not limited to, hyaluronicacid, can be cross-linked, either intramolecularly or intermolecularly,by use of a cross-linking agent. The cross-linking agent can be selectedfrom the group consisting of proanthocyanidin, a bifunctional epoxide, acarbodiimide, and glutaraldehyde, as described above. As anotheralternative, collagen or gelatin can be cross-linked to hyaluronic acid,as described in L.L.H. Huang-Lee & M. E. Nimni, “Fibroblast Contractionof Collagen Matrices With and Without Covalently Bound Hyaluronan,” J.Biomater. Sci. Polymer Ed. 5: 99-109 (1993). As yet another alternative,additional stabilizing cross-links can be introduced into at least oneof the collagen, the elastin, and the glycosaminoglycan, by use of awater-soluble carbodiimide, a periodate, or an epoxide.

Typically, in this method, the mixture is dried, pulverized into smallparticles, suspended in a physiological compatible solution, andinjected subcutaneously or intradermally to humans.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1 is a photomicrograph at ×200 magnification showing the resultswith elastin (without cross-linking) with gelatin as a carrier afterfour weeks in rats treated with the composition (Example 4).

FIG. 2 is a photomicrograph at ×200 magnification showing the resultswith elastin cross-linked with bifunctional epoxide with gelatin as acarrier in rats treated with the composition (Example 4).

FIG. 3 is a photomicrograph at ×200 magnification showing the resultswith elastin cross-linked with proanthocyanidin with gelatin as acarrier in rats treated with the composition (Example 4).

FIG. 4 is a photomicrograph at ×200 magnification showing the resultswith hyaluronic acid cross-linked to a DBM surface, injected with a 23Gneedle in rats treated with the composition (Example 4).

FIG. 5 is another photomicrograph at ×200 magnification showing theresults with hyaluronic acid cross-linked to a DBM surface, injectedwith a 23G needle in rats treated with the composition (Example 4).

FIG. 6 is a photomicrograph at ×200 magnification showing the resultswith DBM cross-linked to hyaluronic acid in rats treated with thecomposition (Example 4).

FIG. 7 shows the results of elastin cross-linked with proanthocyanidin(PA), epoxide (EP), or hyaluronic acid (HA) with subcutaneousimplantation in rats for four weeks. The results are shown in FIG. 7 inthe following arrangement. The leftmost column is elastin cross-linkedwith PA, the center column is elastin cross-linked with EP, and therightmost column is EP cross-linked with HA. The top row shows stainingwith hematoxylin-eosin (H & E); the bottom row shows staining withVerhoeff-Van Gieson stain (VVG), which stains elastic fibers (in black)and collagen (in red). In the results of FIG. 7, the elastin groupsshowed mild foreign tissue responses in all groups. They showed strongtissue regeneration around implants. The overall degradation rates inelastin groups were low. PA and HA appear to induce more infiltration offibroblast cells into elastin particles. Regeneration is obvioussurrounding the elastin particles and into the fiber spaces. In FIG. 7,elastin-PA and elastin-HA are shown at 200× magnification; elastin-EP isshown at 400× magnification.

FIGS. 8A, 8B, and 8C show that the implants kept their injected shapesthroughout the implantation period. Few or no contracture bands are seenin FIGS. 8A, 8B, and 8C (100× magnification for FIG. 8A; 40×magnification for FIG. 8B and FIG. 8C, 4-week implantation). FIG. 8A isa first photomicrograph of an implant after a 4-week implementation at100× magnification showing that the implant retained its shape. FIG. 8Bis a second photomicrograph of an implant after a 4-week implementationat 40× magnification showing that the implant retained its shape. FIG.8C is a third photomicrograph of an implant after a 4-weekimplementation at 40× magnification showing that the implant retainedits shape.

FIG. 9 shows that fibrous tissues formed surrounding the elastinparticles (solid arrow) and fibroblasts migrated into elastin fibers toregenerate fibrous tissues (dotted arrows). Magnification in FIG. 9 was200×.

FIG. 10A, FIG. 10B, FIG. 10C, and FIG. 10D show the results ofimplantation of insoluble collagen together with elastin, elastincross-linked with proanthocyanidin, or elastin alone, using differentimplantation times and routes of implantation. FIG. 10A shows insolublecollagen plus elastin implanted subcutaneously in rats for 8 weeks. FIG.10B shows elastin implanted subcutaneously in rats for 6 weeks (left andright panels). FIG. 10C shows elastin cross-linked with proanthocyanidinimplanted intracutaneously in rats for 6 weeks (left and right panels).FIG. 10D shows elastin cross-linked with hyaluronic acid implantedintracutaneously in rats for 6 weeks (left and right panels).

FIG. 11A and FIG. 11B shows the results with mixtures of collagen andelastin. FIG. 11A shows the results with 75% collagen and 25% elastinimplanted subcutaneously in rats for 4 weeks. FIG. 11B shows the resultswith 25% collagen and 75% elastin implanted subcutaneously in rats for 4weeks.

DETAILED DESCRIPTION OF THE INVENTION

Injectable implant compositions for soft tissue augmentation accordingto the present invention comprise collagen (obtained from skin, tendonor bone) and elastin (obtained from arteries or tendon and ligament).The elastin is the primary filler and is the most important ingredientin achieving the desired soft tissue augmentation effect. Typically thecomposition is suspended in a physiological saline solution forintradermal injection or other soft tissue augmentation. The compositein question is retained at the site of injection for prolonged periodsof time for the purpose of eliminating wrinkles or other visible surfaceimperfections in the skin. Compositions according to the presentinvention can further comprise other ingredients, such as aglycosaminoglycan such as hyaluronic acid. If hyaluronic acid isincluded in the composition, the collagen and hyaluronic acid can form acollagen-hyaluronan complex.

Since the 1980's injectable collagen, usually from bovine sources, butmost recently also of human origin, has been used as a tissue filler toeliminate wrinkles and other facial imperfections, usually associatedwith the aging process. In recent years other natural, synthetic andinert fillers have been used for the same purpose. Presently the use ofthese alternate materials exceeds that of bovine collagen, which isbeing replaced by materials which are more lasting and have a lessertendency to be reactive and induce allergic reactions. Hyaluronic acidand its derivatives, principally cross-linked hyaluronic acid, aregaining more and more acceptance and are becoming the most widely usedtissue fillers. Among these, non-animal derived hyaluronic acid isreceiving the greatest acceptance because of the very small incidence ofallergic reactions and the almost immediate ability to correct facialwrinkles or folds. Most of these acceptable currently used fillers willretain satisfactory beneficial results for periods longer than 6 monthsand up to 1 year, at which time application has to be repeated.

Elastin is an important skin component which contributes to the naturalproperties of skin, and it is one of the dermal components which morereadily becomes depleted during the process of aging. Because of thenature of its chemical composition, and the uniqueness of itscross-linking network, which contributes to make it one of the mostinsoluble proteins in the body, it is very resistant to chemical andenzymatic degradation. Nevertheless UV light has the tendency to destroythe complex cross-links, opening the ring structure of pyridine groupswhich stabilize the protein structure. Sunlight exposure is known toinduce a phenomenon, described as solar elastosis, which causesdegradation and loss of structure of skin elastin and alters theproperties of collagen. The phenomenon is very well described and hasbeen studied extensively in the weathered skin of individuals who areconstantly exposed to sunlight and consequent to sun induced skindamage.

In the present invention we have combined collagen and elastin in theformulation; optionally, a glycosaminoglycan such as hyaluronic acid canbe further included. The natural resistance of elastin to degradation,coupled with its space filling ability, when combined with collagen or acollagen-hyaluronan complex (if hyaluronic acid is present) satisfiesthe criteria of ideal filler. The elastin is the primary filler. Itcontains all the natural materials present in the dermis to achieve theultimate goal of retaining a smooth outer appearance of the skin byeliminating the empty spaces and reversing the contractile process thatis responsible for the superficial uneven appearance of skin.

The present invention contains a mixture of collagen, elastin and,optionally, a glycosaminoglycan such as hyaluronic acid, or any of theircombinations.

Collagen is a fibrous protein with a triple helical structure. A singlemolecule of Type I collagen is comprised of three polypeptide chainswith an aggregate molecular mass of ˜285 kDa. The molecule has a rodlikeshape with a length of ˜3000 Å and a width of ˜14 Å. Collagen has anextremely distinctive amino acid composition; nearly one-third of itsamino acid residues are glycine; another 15% to 30% are proline or4-hydroxyproline (Hyp) residues, with a smaller number of3-hydroxyproline and 5-hydroxylysine residues. These hydroxylated aminoacid residues, which are formed as the result of posttranslationalmodification, confer stability on collagen, probably throughintramolecular hydrogen bonds involving bridging water molecules.Collagen contains many repeated iterations of the sequence Gly-X-Y,where X is often proline and Y is often 4-hydroxyproline. This forms atriple helical structure. Collagen is organized into fibrils. Collagencontains covalently attached carbohydrates in amounts that range from˜0.4% to 12% by weight. The carbohydrates consist mostly of glucose,galactose, and disaccharides, and are covalently attached to collagen atthe 5-hydroxylysine residues. Collagen fibrils are also covalentlycross-linked through reactions involving lysine and histidine residues.

Elastin is another fibrous protein, one which, in its native form, haselastic, rubber-like properties. Elastin is primarily composed of theamino acids glycine, valine, alanine, and proline. It is a specializedprotein with a molecular mass of 64 to 66 kDa. It has an irregular orrandom coil conformation made up of 830 amino acids. Elastin is made bylinking many soluble tropoelastin protein molecules, in a reactioncatalyzed by lysyl oxidase, to make a massive, insoluble, durable arraythat is cross-linked. The amino acid responsible for these cross-linksis lysine. The non-standard amino acids desmosine and isodesmosine areboth found in elastin.

Collagen can be native form, denatured form (gelatin) or cross-linkedendogenously or exogenously.

Collagen can be obtained from a variety of animal and human tissues,such as, but not limited to, skin, tendon, ligament, pericardium, duralmembrane, small intestine mucosa, or bone. In one preferred alternative,collagen is purified from porcine tendon.

Elastin can be obtained from animal and human tendon, such as, but notlimited to, bovine ligamentum nuchae, aorta roots, blood vessels, orpericardium. In one preferred alternative, elastin is purified fromporcine aorta root.

In another preferred alternative, collagen is purified from human bone.Collagen derived from human bone is readily available as a discard frombone banks which manufacture DBM (demineralized bone matrix), usedprimarily as a filler for defects where bone is lost or is insufficientfor adequate repair following surgical procedures (spinal fusion,prosthetic implants, or other procedures).

The DBM which is usually discarded is poor in bone inducing growthfactors but still retains some biological activity which can enhancesoft tissue regeneration. The biological activity of this material, interms of its ability to stimulate fibroblasts to make new collagen atthe site of the implant may further contribute to the efficacy anddurability of the implant by stimulating the local cells of the dermisto make more collagen, therefore supplementing the filling effect of theinjected composite.

Animal studies attached clearly demonstrate the retention of thecollagen-hyaluronic acid-elastin mixture after a prolonged period ofimplantation. Other composites, not cross-linked or without elastin, donot display similar properties which reflect the ability to be retainedand act efficiently as fillers.

Elastin is obtained from tendons and/ligaments of human or animalorigin. It is prepared by sequentially extracting proteoglycans andcollagen from the dissected tissues. (R J Boucek, in Collagen, CRCPress, Editor M. Nimni, vol. III, p 201 (1985). The residue after thisprocess is autoclaved and the insoluble residue consists of pureelastin. It is pulverized for future use.

Collagen and hyaluronic acid are covalently cross-linked using standardcross-linking reagents and procedures (Nimni et al Matrix Biology (1994)14 147-157), U.S. Pat. Nos. 4,378,224 and 5,374,539 to Nimni). Othercross-linking agents are known in the art and are described below. Withregard to cross-linking, the term “intramolecularly” and similarterminology refer to cross-links between the same type of molecule, suchas elastin cross-linked to elastin. The cross-links need not involve thesame molecule, although this is possible. With regard to cross-linking,the term “intermolecularly” and similar terminology refer to cross-linksbetween different types of molecules, such as elastin being cross-linkedto collagen or hyaluronic acid or collagen being cross-linked tohyaluronic acid.

The collagen-HA composite is now suspended in saline together with thepulverized elastin. A rapid-acting local anesthetic is added toeliminate any possible pain associated with the subcutaneous injectionof the suspension. A suitable rapid-acting local anesthetic islidocaine; alternatively, other analogous local anesthetics such asbenzocaine, tetracaine, bupivacaine, cocaine, etidocaine, flecainide,mepivacaine, pramoxine, prilocaine, procaine, chloroprocaine,oxyprocaine, proparacaine, ropivacaine, dyclonine, dibucaine,propoxycaine, chloroxylenol, cinchocaine, dexivacaine, diamocaine,hexylcaine, levobupivacaine, pyrrocaine, risocaine, rodocaine, orpharmaceutically acceptable derivatives and bioisosteres thereof can beused. When the rapid-acting local anesthetic is lidocaine, a suitableconcentration of lidocaine is 0.3%.

Suitable cross-linking reagents and methods include the following: (1)the bioflavonoid proanthocyanidin, a natural cross-linking agent, at0.05% concentration; (2) a bifunctional epoxide such as 1,4-butanedioldiglycidyl ether; (3) a carbodiimide, such as, but not limited to1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and(4) glutaraldehyde. As used herein, the terms “a proanthocyanidin” or“proanthocyanidins” includes all proanthocyanidins and their analoguesas further described in detail below.

Proanthocyanidins are dimers and oligomers of flavan-3-ol units(catechin analogues) linked mainly through C4 to C8 bonds toleucoanthocyanidins. Proanthocyanidins include, but are not limited to,proanthocyanidin, procyanidin (2H-1-benzopyran-3,4,5,7-tetrol,2-(3,4-dihydroxyphenyl)-2-(2-(3,4-dihydroxyphenyl)-3,4-dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl)oxy)-3,4-dihydro),procyanidin B, procyanidin B2, rhatannin, procyanidol oligomer,procyanidin C, procyanidin B3, procyanidin B1, selligueain A(8,14-methano-2H,14H-1-benzopyrano(7,8-d)(1,3)benzodioxocin-3,5,11,13,15-pentol,4-(3,4-dihydro-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-2H-1-benzopyran-8-yl)-3,4-dihydro-2,8-bis(4-hydroxyphenyl)-,(2R-(2α,3α,4β(2R*,3S*),8β,14β,15R*)),geranin A, geranin D, procyanidin B5, procyanidin B5-3′-O-gallate,vitisinol, amurensisin, terminalin, geranin B, 6,8-dihydroxyafzelin,afzelin-3″-O-gallate, geranin C, afzelin, flavangenol, carallidin,mahuannin A, proanthocyanidin A1, proanthocyanidin A2, procyanidin D,and analogues, derivatives, and bioisosteres of these compounds.

As described further below, it is highly preferred to cross-link theelastin with a proanthocyanidin. Preferably, a proanthocyanidin is usedat a concentration of from about 0.01% to about 1.0%; more preferably,the proanthocyanidin is used at a concentration of from about 0.1% toabout 1.0%. The pH is preferably from about 5.5 to about 8.4; morepreferably, the pH is from about 7.8 to about 8.4. The interaction ofthe proanthocyanidin can be described as stabilizing, cross-linking, orcoating. The use of a proanthocyanidin to cross-link, stabilize, or coatelastin in a microparticle suspension is highly preferred in order toprevent its biodegradation. If not cross-linked, stabilized, or coated,elastin degrades rapidly, as determined by an in vitro enzymedegradation test.

Other carbodiim ides are known in the art, including, but not limited todicyclohexylcarbodiimide (DCC), 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide (EAC);1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl-carbodiimidemetho-p-toluenesulfonate (CMC), andN-benzyl-N′-3-dimethylaminopropyl-carbodiimide4 hydrochloride (BDC).These and other suitable cross-linking reagents and procedures aredescribed in S.S. Wong, “Chemistry of Protein Conjugation andCross-linking” (CRC Press, Boca Raton, Fla., 1993). Still other suitablecross-linking agents can be used, such as the difunctionally activatedpolyethylene glycols described in U.S. Pat. No. 5,643,464 to Rhee et al.

Another cross-linking method is oxidative cross-linking with periodate.When periodate is used as a cross-linker, the aldehydes formed can thenbe stabilized further by reaction with sodium borohydride or sodiumcyanoborohydride to give rise to more permanent cross-links, such ascollagen-hyaluronic acid cross-links, as shown in FIG. 5 of L.L.H.Huang-Lee & M.E. Nimni, “Fibroblast Contraction of Collagen MatricesWith and Without Covalently Bound Hyaluronan,” J. Biomater. Sci. PolymerEd. 5: 99-109 (1993). The reaction with sodium borohydride or sodiumcyanoborohydride results in the reduction of aldehydes to alcohols.Additionally, periodate can modify the vicinal hydroxyl groups inhyaluronic acid to aldehyde groups. Free amino groups in collagen thencan form Schiff bases with these aldehyde groups. These Schiff bases canalso be reduced by reaction with sodium borohydride or sodiumcyanoborohydride to more stable amides.

As another alternative, when cross-linking is performed withglutaraldehyde, a calcification inhibitor can be used. Suchcalcification inhibitors are described in U.S. Pat. No. 4,378,224 toNimni et al. Additionally, bridging agents, such as diamines, can befurther included as described in U.S. Pat. No. 4,378,224 to Nimni et al.Suitable diamines are aliphatic diamines, including, but not limited to,hexanediamine.

Typically, compositions according to the present invention areformulated for injection. Typically, the route of injection issubcutaneous or intradermal, although other routes of injection arepossible. Formulations suitable for injection are well known in the art,and can, for example, include sterile aqueous solutions and/ordispersions; formulations including sesame oil, peanut oil, syntheticfatty acid esters such as ethyl oleate, triglycerides, and/or aqueouspropylene glycol; and/or sterile powders for the extemporaneouspreparation of sterile injectable solutions and/or dispersions. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutionsor suspensions where appropriate. In all cases the form must be sterileand/or must be fluid to the extent that the solution will pass readilythrough a syringe and needle of suitable diameter for administration. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms, such asbacteria or fungi. Other additives, excipients, and preservatives wellknown in the art can be used.

Accordingly, one aspect of the invention is an injectable compositionhaving dermal filling and tissue expanding activity comprising:

(1) a quantity of elastin sufficient to bring about dermal filling andtissue expansion when injected into a subject in need of dermal fillingand tissue expansion; and

(2) a pharmaceutically acceptable carrier.

Typically, the elastin is selected from the group consisting ofnon-human mammalian elastin and human elastin. If the elastin isnon-human mammalian elastin, it is typically porcine elastin.

The pharmaceutically acceptable carrier can comprise gelatin. It canfurther comprise phosphate buffered saline or other excipients; otherexcipients such as glucose or maltose have been described and are knownin the art. In another alternative, the pharmaceutically acceptablecarrier can comprise carboxymethylcellulose.

In one preferred alternative, the composition further comprises arapid-acting local anesthetic. The rapid-acting local anesthetic can beselected from the group consisting of lidocaine, benzocaine, tetracaine,bupivacaine, cocaine, etidocaine, flecainide, mepivacaine, pramoxine,prilocaine, procaine, chloroprocaine, oxyprocaine, proparacaine,ropivacaine, dyclonine, dibucaine, propoxycaine, chloroxylenol,cinchocaine, dexivacaine, diamocaine, hexylcaine, levobupivacaine,pyrrocaine, risocaine, rodocaine, and pharmaceutically acceptablederivatives and bioisosteres thereof. Typically, the rapid-acting localanesthetic is lidocaine.

The elastin can be cross-linked with a cross-linking agent, as describedabove. The cross-linking agent can be selected from the group consistingof a proanthocyanidin, a bifunctional epoxide, a carbodiimide, andglutaraldehyde. If the cross-linking agent is a bifunctional epoxide, itis typically 1,4-butanediol diglycidyl ether. If the cross-linking agentis a carbodiimide, the carbodiimide is typically selected from the groupconsisting of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC), dicyclohexylcarbodiimide (DCC),1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide iodide (EAC);1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl-carbodiimidemetho-p-toluenesulfonate (CMC), andN-benzyl-N′-3-dimethylaminopropyl-carbodiimide hydrochloride (BDC).Preferably, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

In one preferred alternative, the elastin is cross-linked with aglycosaminoglycan. The glycosaminoglycan can be selected from the groupconsisting of hyaluronic acid, chondroitin sulfate, pentosanpolysulfate, dermatan sulfates, heparin, heparan sulfates, and keratansulfates. Typically, the glycosaminoglycan is hyaluronic acid. Suitablecross-linking agents are as described above. When the glycosaminoglycanis hyaluronic acid, the hyaluronic acid can be obtained from roostercombs or can be synthesized by bacteria.

In another preferred alternative, the composition further comprisescollagen. The collagen can be native or denatured collagen obtained fromskin, tendon, ligament, pericardium, dural membrane, small intestinemucosa or bone. The collagen can be used in soluble form, fibrillarform, a form of an insoluble slurry, or hydrogel form. The collagen canbe human Type I collagen. The human Type I collagen can be obtained fromdemineralized bone matrix (DBM). Alternatively, the collagen can becollagen from porcine tendon. The collagen can be cross-linkedintramolecularly. Suitable cross-linking agents are described above.Alternatively, the collagen can be cross-linked to the elastin, or thecollagen can be cross-linked to a glycosaminoglycan such as, but notlimited to, hyaluronic acid, which can also be included in thecomposition in that alternative.

The elastin can be obtained from human or animal ligaments or arteries.Typically, the elastin is purified from non-elastineous proteins andproteoglycans. Typically, the elastin is cryofractured into a fineparticle. Preferably, the size of the fine particle is from about 50 μmto about 210 μm. Typically, the elastin concentration is from about 20mg/mL to about 300 mg/mL; preferably, the elastin concentration is fromabout 20 mg/mL to about 100 mg/mL.

In one alternative of this aspect of the invention, the compositionfurther comprises collagen and a glycosaminoglycan such as, but notlimited to, hyaluronic acid, and wherein one or more of the elastin, thecollagen, and the glycosaminoglycan are cross-linked, eitherintramolecularly or intermolecularly. Suitable cross-linking agents aredescribed above.

Another aspect of the invention is a method of dermal filling and tissueexpansion comprising the step of injecting a quantity of this embodimentof a composition according to the present invention as described abovesufficient to induce dermal filling and tissue expansion to a patient inneed thereof. In this method, the dosage, the frequency of the dosage,and the route of injection can be chosen by a skilled clinician takinginto account such factors as the condition of the skin of the patient,the specific wrinkles, folds, or other imperfections visible, the degreeof improvement desired, the sex, age, weight, and general physicalcondition of the patient, the presence of other preexisting conditionsaffecting the skin or the connective tissue, the possibility of sideeffects or hypersensitivity caused by any ingredient of the composition,and the existence of conditions, such as liver or kidney conditions,that will affect the pharmacokinetics of any ingredient of thecomposition.

Another aspect of the present invention is an injectable compositionhaving dermal filling and tissue expanding activity comprising:

(1) an insoluble collagen suspension in a quantity sufficient to bringabout dermal filling and tissue expansion when injected into a subjectin need of dermal filling and tissue expansion; and

(2) a pharmaceutically acceptable carrier.

Typically, in this composition, the concentration of collagen is fromabout 20 mg/mL to about 100 mg/mL. Typically, in this composition, thecollagen is cross-linked with a cross-linking agent, as described above.

Typically, in this composition, the pharmaceutically acceptable carrieris gelatin.

In this aspect of the invention, the composition can further compriseelastin. In one preferred alternative when the composition compriseselastin, the composition comprises 75 mg of elastin particles and 25 mgof insoluble collagen per 1.0 mL of pharmaceutically acceptable carrier.In this alternative, the pharmaceutically acceptable carrier can begelatin.

In this aspect of the invention, the collagen can be partially denaturedinto gelatin to aid in maintaining the components in suspension and infacilitating the injection of the mixture through a narrow gauge needle.

Other components can be included. For example, the composition canfurther comprise a growth factor. The growth factor can be one or moreof the following: an interleukin; a bone morphogenetic protein (BMP);brain-derived neurotrophic factor (BDNF); transforming growth factor α(TGF α); transforming growth factor β₁ (TGF β₁); transforming growthfactor β₂ (TGF β₂); acidic fibroblast growth factor (aFGF); basicfibroblast growth factor (bFGF); granulocyte colony-stimulating factor(G-CSF); glial cell line-derived growth factor (GDNF);granulocyte/macrophage colony-stimulating factor (GM-CSF); growthhormone; haemoinfiltrate CC chemokine 1 (HCC-1); insulin-like growthfactor I (IGF I); insulin-like growth factor II (IGF II); macrophagecolony-stimulating factor (M-CSF); and stem cell factor (SCF). The useof other growth factors is possible.

These growth factors are described in general in K. A. Fitzgerald etal., The Cytokine Facts Book (2d ed., Academic Press, San Diego, 2001).Specifically, the interleukins include IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL12, IL-13, IL-14, IL-15, IL-16,IL-17, and IL-18. IL-1 exists in two forms, IL-1α and IL-1β. IL-1α has159 amino acids in the mature form in human and 156 in mouse; IL-1β has153 amino acids in the mature form in human and 159 in mouse. IL-2 has133 amino acids in the mature form in human and 149 in mouse. IL-3 has133 amino acids in the mature form in human and 140 in mouse. IL-4 has129 amino acids in the mature form in human and 120 in mouse. IL-5 has115 amino acids in the mature form in human and 113 amino acids inmouse. IL-6 has 183 amino acids in the mature form in human and 187 inmouse. IL-7 has 152 amino acids in the mature form in human and 129 inmouse. IL-8 has 99 amino acids in the mature form in human. IL-9 has 126amino acids in the mature form in human and 126 in mouse. IL-10 has 160amino acids in the mature form in human and 160 in mouse. IL-11 has 178amino acids in the mature form in human and 178 in mouse. IL-12 has 115amino acids in the mature form in human and 113 amino in mouse. IL-13has 112 amino acids in the mature form in human and 113 amino in mouse.IL-114 has 483 amino acids in the mature form in human. IL-15 has 113amino acids in the mature form in human and 114 in mouse. IL-16 has 115amino acids in the mature form in human and 113 amino in mouse. IL-17has 132 amino acids in the mature form in human and 133 amino in mouse.IL-18 has 522 amino acids in the mature form in human and 519 in mouse.

The BMPs are described in further detail in the following publications:(1) F. P. Luyten et al., “Purification and Partial Amino Acid Sequenceof Osteogenin, a Protein Initiating Bone Differentiation,” J. Biol.Chem. 264: 13377-13380 (1989); (2) E. Özkaynak et al., “MurineOsteogenic Protein (OP-1): High Levels of mRNA in Kidney,” Biochem.Biophys. Res. Commun. 179: 116-123 (1991); (3) R. M. Harland et al.,“The Transforming Growth Factor β Family and Induction of the VertebrateMesoderm: Bone Morphogenetic Proteins are Ventral Inducers,” Proc. Natl.Acad. Sci. USA 91: 10243-10246 (1994); (4) S. K. Maiti & G. R. Singh,“Bone Morphogenetic Proteins-Novel Regulators of Bone Formation,” Ind.J. Exp. Biol. 36: 237-244 (1998); (5) J. M. Wozney et al., “NovelRegulators of Bone Formation: Molecular Clones and Activities,” Science242: 1528-1534 (1988); (6) D. M. Kingsley et al., “What Do BMPs Do inMammals? Clues from the Mouse Short-Ear Mutation,” Trends Genet. 10:16-21 (1994); (7) C. Scheufler et al., “Crystal Structure of Human BoneMorphogenetic Protein-2 at 2.7 A Resolution,” J. Mol. Biol. 287: 103-115(1999); (8) J. Q. Feng et al., “Structure and Sequence of Mouse BoneMorphogenetic Protein-2 Gene (BMP-2): Comparison of the Structures andPromoter Regions of BMP-2 and BMP-4 Genes,” Biochim. Biophys. Acta 1218:221-224 (1994); (9) N. Ghosh-Choudhury et al., “Expression of the BMP 2Gene During Bone Cell Differentiation,” Crit. Rev. Eukaryot. Gene Expr.4: 345-355 (1994); (10) B. L. Rosenzweig et al., “Cloning andCharacterization of a Human Type II Receptor for Bone MorphogeneticProteins,” Proc. Natl. Acad. Sci. USA 92: 7632-7636; (11) L. J. Jonk etal., “Identification and Functional Characterization of a Smad BindingElement (SBE) in the JunB Promoter That Acts as a Transforming GrowthFactor-β, Activin, and Bone-Morphogenetic-Protein-Inducible Enhancer,”J. Biol. Chem. 273: 21145-21152 (1998); and (12) M. Kawabata et al.,“Signal Transduction by Bone Morphogenetic Proteins,” Cytokine GrowthFactor Rev. 9: 49-61 (1998). The BMPs represent a family of proteinsthat initiate, promote, and maintain cartilage and bone morphogenesis,differentiation and regeneration in both the developing embryo and theadult. There are more than 30 known BMPs, of which 15 are found inmammals. BMPs belong to the transforming growth factor β (TGFβ)superfamily, which includes TGFβs, activins/inhibins,Mullerian-inhibiting substance (MIS) and glial cell line-derivedneurotrophic factor. Comparison and alignment of the amino acidsequences of BMPs reveal that BMPs, except for BMP-1, share a commonstructural motif that is distinct from the structure of BMP-1. TheseBMPs include BMP-2, BMP-3, BMP-3b, BMP4, BMP-5, BMP-6, BMP-7, BMP-8,BMP-8B, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF9, GDF-10, and nodal. Inthis specification, the term “BMP,” without further qualification, is tobe taken to include BMP-1; the term “BMP sharing a common structuralmotif” is to be taken to include BMPs other than BMP-1 and excludeBMP-1. These BMPs sharing a common structural motif are disulfide-linkeddimeric proteins. BMP-1 is not properly a BMP family member; rather itis a procollagen C proteinase related to Drosophila tolloid and which ispostulated to regulate BMP activity through proteolysis of BMPantagonists/binding proteins.

BDNF has 119 amino acids in the mature form in both human and, mouse. Ithas 70% β3-sheet and is expressed as a tightly associated homodimer.

TGF α has 50 amino acids in the mature form in both human and rat. It isa small integral membrane protein.

TGF β₁ has 112 amino acids in the mature form in humans; there isgreater than 98% homology between the functional regions of human andmouse molecules.

TGF β₂ also has 112 amino acids in the mature form in humans; there isagain greater than 98% homology between the functional regions of humanand mouse molecules.

The growth factor aFGF has 155 amino acids in the mature form in humansand in mice.

The growth factor bFGF has 155 amino acids in the mature form in humansand 154 amino acids in the mature form in mice. The growth factor bFGFis composed entirely of a β-sheet structure with a 3-fold repeat of afour-stranded antiparallel β-meander that forms a barrel-like structurewith three loops.

G-CSF has 177 or 174 amino acids in the mature form in humans and 178amino acids in the mature form in mice. The 177-amino-acid and174-amino-acid forms in humans are alternatively spliced variants. G-CSFforms a four a-helical bundle structure.

GDNF has 134 amino acids in the mature form in humans and mice.

GM-CSF has 127 amino acids in the mature form in humans and 124 aminoacids in the mature form in mice. The molecule comprises a two-strandedantiparallel β-sheet with an open bundle of four α-helices.

Growth hormone has 191 amino acids in the mature form in humans and 190amino acids in the mature form in mice. The molecule forms a fourα-helical bundle structure.

HCC-1 has 74 amino acids in the mature form in humans.

IGF I exists in two isoforms, IGF IA and IGF IB. Both consist of A and Bdomains, homologous to the A and B chains of insulin, connected by a Cpeptide and an eight-amino-acid extension at the C terminus termed the Ddomain. In humans and in mice, the IGF IA and IGF IB isoforms are both70 amino acids in length in the mature form. IGF I has athree-dimensional structure similar to insulin.

IGF II has 67 amino acids in the mature form in both humans and mice. Italso consists of A, B, C, and D domains. IGF II also has athree-dimensional structure similar to insulin.

M-CSF exists in three mature forms in humans, with 522, 406, and 224amino acids respectively. In mice, the mature form of M-CSF has 519amino acids. At least a portion of the structure of M-CSF comprises twobundles of four a-helices laid end to end.

SCF has 248 or 220 amino acids in the mature form in both humans andmice, existing in long and short membrane-bound forms after removal ofthe predicted signal peptide. The molecule exists as a noncovalentlylinked homodimer that contains extensive α-helical and β-pleated sheets.

These growth factors can exist in multiple forms, such as: (1) splicingvariants produced from mRNAs generated by spicing from alternativesites; (2) variants produced by proteolysis, such as the cleavage ofsignal peptides or propeptides; (3) variants produced by the presence orlack of glycosylation, typically N-linked glycosylation; (4)naturally-occurring isoforms; (5) naturally-occurring mutations orallelic variants; and (6) artificial variants produced by geneticengineering in which one or more amino acids in the primary sequence arealtered by techniques such as site-specific mutagenesis; such artificialvariants are frequently designated muteins. In general, these multipleforms are within the scope of the present invention when they exist orcan be produced for a particular growth factor.

Another aspect of the invention is a method of dermal filling and tissueexpansion comprising the step of injecting a quantity of this embodimentof a composition according to the present invention as described abovesufficient to induce dermal filling and tissue expansion to a patient inneed thereof. In this method, the dosage, the frequency of the dosage,and the route of injection can be chosen by a skilled clinician takinginto account the factors described above.

Yet another aspect of the present invention is a method for augmentingthe volume area of dermal tissue at sites where wrinkles and othertissue imperfections have caused visible changes associated with loss orrearrangement of the subcutaneous collagen network comprising the stepof: providing an effective amount of a composition, which can beinjected locally, and which consists of a mixture of macromoleculeswhich are native constituents of the skin, the mixture comprisingcollagen, elastin and, optionally, a glycosaminoglycan, such as, but notlimited to, hyaluronic acid, which are held together by covalent, ionicor hydrogen bonds, and which include natural materials which have beendemonstrated to increase the natural synthesis of these molecules bycells which reside in the dermis. In this method, the collagen can benative or denatured collagen obtained from skin, tendon, ligament,pericardium, dural membrane, small intestine mucosa or bone. Thecollagen can be used in soluble form, fibrillar form, a form of aninsoluble slurry, or hydrogel form. The collagen can be cross-linkedintramolecularly. Alternatively, the collagen can be cross-linked to theelastin or to the glycosaminoglycan, such as, but not limited to,hyaluronic acid. The collagen can be insoluble collagen; when thecollagen is insoluble collagen, the concentration of insoluble collagenis typically from about 20 mg/mL to about 100 mg/mL.

The elastin used can be obtained from human or animal ligaments orarteries. The elastin is typically purified from non-elastineousproteins and proteoglycans. The elastin is typically cryofractured intoa fine particle. Typically, the size of the fine particle is from about50 μm to about 210 μm.

In this method, the injectable composition used typically comprisesgelatin.

In this method, typically, the elastin concentration is from about 20mg/mL to about 100 mg/mL.

In this method, when the glycosaminoglycan is hyaluronic acid, thehyaluronic acid used can be obtained from rooster combs or issynthesized by bacteria.

In this method, at least one of the collagen, the elastin, and theglycosaminoglycan, such as, but not limited to, hyaluronic acid, can becross-linked, either intramolecularly or intermolecularly, by use of across-linking agent. The cross-linking agent can be selected from thegroup consisting of proanthocyanidin, a bifunctional epoxide, acarbodiimide, and glutaraldehyde, as described above. As anotheralternative, additional stabilizing cross-links can be introduced intoat least one of the collagen, the elastin, and the glycosaminoglycan, byuse of a water-soluble carbodiimide, a periodate, or an epoxide.Specifically, proanthocyanidin as a cross-linker acts as a protector ofelastin against ultraviolet degradation. Additionally, proanthocyanidincombined with a gelatin or collagen-hyaluronic acid matrix can inhibitmatrix degradation and contraction by fibroblasts as described in L. L.H. Huang-Lee & M. E. Nimni, “Fibroblast Contraction of Collagen MatricesWith and Without Covalently Bound Hyaluronan,” J. Biomater. Sci. PolymerEd. 5: 99-109 (1993). Additionally, when periodate is used as across-linker, the aldehydes formed can then be stabilized further byreaction with sodium borohydride to give rise to more permanentcross-links, such as collagen-hyaluronic acid cross-links, as shown inFIG. 5 of L. L. H. Huang-Lee & M. E. Nimni, “Fibroblast Contraction ofCollagen Matrices With and Without Covalently Bound Hyaluronan,” J.Biomater. Sci. Polymer Ed. 5: 99-109 (1993). The reaction with sodiumborohydride results in the reduction of the aldehyde groups to alcohols.As another alternative, when cross-linking is performed withglutaraldehyde, a calcification inhibitor can be used. Suchcalcification inhibitors are described in U.S. Pat. No. 4,378,224 toNimni et al. Additionally, bridging agents, such as diamines, can befurther included as described in U.S. Pat. No. 4,378,224 to Nimni et al.Suitable diamines are aliphatic diamines, including, but not limited to,hexanediamine.

Typically, in this method, the mixture is dried, pulverized into smallparticles, suspended in a physiological compatible solution, andinjected subcutaneously or intradermally to humans.

The following references are relevant to the understanding of thepresent invention, although Applicants do not state that such referencesconstitute relevant prior art: (1) Injectable collagen for tissueaugmentation, D G Wallace et al., In Collagen, Vol III, Editor M. E.Nimni, CRC Press (1988), pp 117-144; (2) U.S. Pat. No. 4,233,360 to Lucket al.; and (3) U.S. Pat. No. 4,424,208 to Wallace et al.

Additionally, the following patents are relevant to the understanding ofthe present invention and can provide alternative methods and reagentsfor practicing the invention, although Applicants do not state that suchreferences constitute relevant prior art: U.S. Pat. No. 4,557,664 toChu; U.S. Pat.t No. 4,582,640 to Smestad et al.; U.S. Pat. No. 4,760,131to Sundsmo et al.; U.S. Pat. No. 4,774,227 to Piez et al.; U.S. Pat. No.4,789,663 to Wallace et al.; U.S. Pat. No. 4,803,075 to Wallace et al.;U.S. Pat. No. 4,863,732 to Nathan et al.; U.S. Pat. No. 4,950,483 toKsander et al., U.S. Pat. No. 5,110,604 to Chu et al.; U.S. Pat. No.5,162,430 to Rhee et al.; U.S. Pat. No. 5,219,576 to Chu et al.; U.S.Pat. No. 5,304,595 to Rhee et al.; U.S. Pat. No. 5,328,955 to Rhee etal.; U.S. Pat. No. 5,413,791 to Rhee et al.; U.S. Pat. No. 5,446,091 toRhee et al.; U.S. Pat. No. 5,470,911 to Rhee et al.; U.S. Pat. No.5,475,052 to Rhee et al.; U.S. Pat. No.5,476,666 to Rhee et al.; U.S.Pat. No. 5,510,418 to Rhee et al.; U.S. Pat. No. 5,510,418; U.S. Pat.No. 5,523,348 to Rhee et al.; U.S. Pat. No. 5,527,856 to Rhee et al.;U.S. Pat. No. 5,550,187 to Rhee et al.; and U.S. Pat. No. 5,614,587 toRhee et al.

Applicants believe that the novelty of the present invention resides inthe following: (1) the combination of all 3 ingredients, collagen,elastin and HA; (2) the use of the most insoluble form of tissuecollagen, namely bone collagen, in one of our preferred embodiments; (3)cross-linking with a proanthocyanidin, a natural bioflavonoid; (4) useof this same bioflavonoid to enhance new collagen synthesis (as shown inour paper of 2003 (Han B Nimni et al. Proanthocyanidin: a naturalcross-linking reagent for stabilizing collagen matrices, J. Biomed.Mater. Res 65 (1): 118-124) and to protect elastin against UV damage;(5) the suspension of particulate elastin in a mixture ofcollagen-gelatin-hyaluronic acid cross-linked with proanthocyanidin(with or without further enhancement of cross-links by carbodiimide orepoxide reagents); (6) the long term persistence of this mixture belowthe dermal epidermal junction; (7) the negligible or minimal immunereactivity of this composite; (8) the preferred embodiments use pigtendon, which has significant identity to human collagen, and also usedemineralized bone matrix from various sources, including, but notlimited to, human bone obtained from bone banks. The use of aproanthocyanidin to cross-link, stabilize, or coat elastin in amicroparticle suspension is highly preferred in order to prevent itsbiodegradation. If not cross-linked, stabilized, or coated, elastindegrades rapidly, as determined by an in vitro enzyme degradation test.

The invention is illustrated by the following examples. These examplesare for illustrative purposes only, and are not intended to limit theinvention.

EXAMPLES Example 1 Elastin Preparation

Porcine aorta roots weighing about 300 g was soaked in PBS overnight at4° C. for 24 hours with 3 changes of solution to remove adherent bloodand other water soluble proteins. After wash, aorta roots were soaked in1000 mL of 0.4 M guanidine HCl and heated at 100° C. for 45 min. Afterbeing rinsed with water, the materials were transferred into 500 mL of0.1 N NaOH and heated at 50° C. for 45 min. After subsequent washing,tissues were air-dried and ready for cryofracture.

Alternatively, after initial washing with PBS, aorta roots were soakedin 0.5 N NaOH and heated at 50° C. for 45 min. Samples were washed andthen sonicated in water for 30 min. Tissues were heated at 100° C. inwater for 30 min followed by water washing (three times). Elastin wasair-dried in fume hood followed by cryofracture (pulverization) inliquid nitrogen (SPEX Freezer Mill). Particles were sieved and particlesized from 50-210 μm were collected.

Example 2 Collagen Purification

After removing attached tissues, porcine tendon was washed with PBS at4° C. for 12 hours with three changes of solution. After tissue wassectioned into fine slices, tissues were delipidated withchloroform/methanol overnight at 4° C. Tissues were then digested in 0.5M acetic acid with 0.5 mg/ml pepsin (Sigma) for 48 hours at 4° C. toremove telopeptide. The supernatants were collected aftercentrifugation. Collagen was precipitated with 2.5 M NaCl. Theprecipitated collagen was then dissolved in 1 M NaCl, 0.05 M Tris, pH7.4, then sequentially dissolved and precipitated with 1.8 M NaCl and2.5 M NaCl to produce pure Type I collagen. Collagen was dissolved into0.5 M acetic acid and further dialyzed against 0.01 N HCl. Purifiedcollagen was stored at 4° C.

To obtain denatured collagen, collagen was neutralized with 0.1 N NaOHto pH 7.4. Collagen solution was autoclaved at 127° C. for 30 minutes todenature the collagen.

Alternatively, insoluble bone collagen was used. Human DBM(demineralized bone matrix) obtained from tissue bank with a particlesize from 50-210 μm was also used as insoluble collagen source. DBM wasinactivated with 6M Guanidine-HCl for 24 hours at 4° C. The materialafter processing is sterile and devoid of any viral pathogens andantigenic proteins for the respective species for which it will beutilized.

Example 3 Injectable Formulations

The following injectable formulations were prepared:

Formulation I comprises elastin cross-linked with proanthocyanidin (PA)with gelatin as a carrier. Elastin was further cross-linked with 0.05%proanthocyanidin solution overnight, after washing with PBS three times.250 mg elastin particles were suspended in 3 mL of 10% porcine tendongelatin solution.

Formulation II comprises elastin cross-linked with bifunctional epoxidewith gelatin as a carrier. Elastin was added into 4% EGDA, 4 M NaCl in0.5 N NaOH for 2 h at 37° C., and then transferred into PBS solution andwashed for 2 hours. 250 mg elastin particles were suspended in 3 mL of10% porcine tendon gelatin solution.

Formulation III comprises elastin cross-linked with hyaluronic acid (HA)with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)and gelatin as a carrier. Cross-linked HA was prepared by first adding29.72 mg EDC and 53.6 mg NHS in 10 mL deionized water. 100 mg HA(rooster comb) was added to the EDC/NHS solution and stirred for 2hours. Elastin was added to the cross-linked HA solution overnight. Theparticles were washed with PBS three times. Elastin-HA particles weresuspended in porcine tendon gelatin solution for injection.

Formulation IV comprises a suspension of elastin, insoluble collagen,and gelatin. 75 mg of elastin particles and 25 mg of insoluble bonecollagen were mixed and suspended in 1.0 mL of gelatin solution.

Formulation V comprises insoluble bone collagen cross-linked withhyaluronic acid and gelatin as a carrier, with or without elastin. Inthis formulation, in the activated HA EDC/NHS solution described above,insoluble bone collagen particles were stirred overnight. Insoluble bonecollagen particles with or without 25 mg of elastin were then suspendedin gelatin solution for injection.

Example 4 Animal Tests

Fisher 344 rats were anesthetized with intraperitoneal injections ofxylazine (10 mg/kg) and ketamine (50 mg/kg). Skin was shaved on thedorsal site and 23-27 G needle was used to inject materials intosubcutaneous sites. Each animal received 4 injections. After 2 weeks or4 weeks, animals were sacrificed and injected samples and surroundingtissues were evaluated macroscopically and microscopically afterhistological processing.

The results are shown in FIGS. 1-11, below. It can be clearly seen thatthe mixtures were in general well-tolerated, with differences inresponse and persistence of the implants depending on the ingredientsand modalities of handling. In general, elastin that was notcross-linked undergoes partial degradation. When cross-linked withepoxide, it is tolerated extremely well. In general, results wherecross-linking was performed with proanthocyanidin were similar, withincreased persistence and a variable degree of inflammatory responsedepending on the concentration of proanthocyanidin. The moderateinflammatory response is viewed as an advantage, as it dissipates after4 weeks, but results in an enhanced deposition of collagen in the areaof the implant.

This deposition of collagen, which seems to correlate with the rate ofresorption of the implant, extends the space-filling effect of theimplant, thus contributing to the efficacy of the procedure. It isexpected that this biological response will correlate with the long termelimination of wrinkles and other undesirable skin surfacecharacteristics.

FIG. 1 shows the results with elastin (without cross-linking) withgelatin as a carrier after four weeks (×200 magnification for FIGS.1-6).

FIG. 2 shows the results with elastin cross-linked with bifunctionalepoxide with gelatin as a carrier.

FIG. 3 shows the results with elastin cross-linked with proanthocyanidinwith gelatin as a carrier.

FIG. 4 shows the results with hyaluronic acid cross-linked to a DBMsurface, injected with a 23G needle.

FIG. 5 shows the results with hyaluronic acid cross-linked to a DBMsurface, injected with a 23G needle.

FIG. 6 shows the results with DBM cross-linked to hyaluronic acid.

FIG. 7 shows the results of elastin cross-linked with proanthocyanidin(PA), epoxide (EP), or hyaluronic acid (HA) with subcutaneousimplantation in rats for four weeks. The results are shown in FIG. 7 inthe following arrangement. The leftmost column is elastin cross-linkedwith PA, the center column is elastin cross-linked with EP, and therightmost column is EP cross-linked with HA. The top row shows stainingwith hematoxylin-eosin (H & E); the bottom row shows staining withVerhoeff-Van Gieson stain (VVG), which stains elastic fibers (in black)and collagen (in red). In the results of FIG. 7, the elastin groupsshowed mild foreign tissue responses in all groups. They showed strongtissue regeneration around implants. The overall degradation rates inelastin groups were low. PA and HA appear to induce more infiltration offibroblast cells into elastin particles. Regeneration is obvioussurrounding the elastin particles and into the fiber spaces. In FIG. 7,elastin-PA and elastin-HA are shown at 200× magnification; elastin-EP isshown at 400× magnification. In these experiments, both intermolecularand intramolecular cross-links were formed by the proanthocyanidin orepoxide when these reagents were used to cross-link elastin.Glutaraldehyde cannot be used to cross-link elastin because elastinlacks free ε-NH₂ groups for such cross-linking by reaction with acarbonyl group. If hyaluronic acid was used, carbodiimide was used asthe cross-linking reagent to cross-link hyaluronic acid to elastin,forming intermolecular cross-links. In these and the followingexperiments, the elastin concentration was from 70 mg/mL to 320 mg/mL.In one embodiment, 78 mg/mL elastin in 5% gelatin/PBS was used ascarrier. Alternatively, carboxymethylcellulose was used as a carrier.

FIGS. 8A, 8B, and 8C show that the implants kept their injected shapesthroughout the implantation period. Few or no contracture bands are seenin FIG. 8A, FIG. 8B, and FIG. 8C (100× magnification, 4-weekimplantation). In FIG. 8A, FIG. 8B, and FIG. 8C, and other similarfigures, a blue-black color was seen as the result of specific stainingof elastin by the Verhoeff-Van Gieson staining method. Using theVerhoeff-Van Gieson staining method, staining was performed as follows:After slides were deparaffinized and hydrated with distilled water,slides were first stained 15 minutes in Verhoeff's Iron Hematoxylin.Excess stain was rinsed off in tap water for 20 minutes. Solution of 2%aqueous ferric chloride was used to differentiate elastic fibers bystaining these fibers black. Then slides were placed in 5% sodiumthiosulfate for 1-2 minutes and counterstained in Van Gieson solutionfor 1 minute. Elastic fibers were stained intensely blue to black.Collagen was stained pink to red and other tissue elements were stainedyellow. The smaller panels (FIG. 8B and FIG. 8C) were with 40×magnification. They are different sections from the same type ofimplants, but not the same tissue. These panels show that the injectedsamples are stay at the injection place and keep their original shapes.FIG. 8A is a first photomicrograph of an implant after a 4-weekimplementation at 100× magnification showing that the implant retainedits shape. FIG. 8B is a second photomicrograph of an implant after a4-week implementation at 40× magnification showing that the implantretained its shape. FIG. 8C is a third photomicrograph of an implantafter a 4-week implementation at 40× magnification showing that theimplant retained its shape.

FIG. 9 shows that fibrous tissues formed surrounding the elastinparticles (solid arrow) and fibroblasts migrated into elastin fibers toregenerate fibrous tissues (dotted arrows). Magnification in FIG. 9 was200×.

FIG. 10A, FIG. 10B, FIG. 10C, and FIG. 10D show the results ofimplantation of insoluble collagen together with elastin, elastincross-linked with proanthocyanidin, or elastin alone, using differentimplantation times and routes of implantation. FIG. 10A shows insolublecollagen plus elastin implanted subcutaneously in rats for 8 weeks. FIG.10B shows elastin implanted subcutaneously in rats for 6 weeks (left andright panels). FIG. 10C shows elastin cross-linked with proanthocyanidinimplanted intracutaneously in rats for 6 weeks (left and right panels).FIG. 10D shows elastin cross-linked with hyaluronic acid implantedintracutaneously in rats for 6 weeks (left and right panels).

FIG. 11A and FIG. 11B show the results with mixtures of collagen andelastin. FIG. 11A shows the results with 75% collagen and 25% elastinimplanted subcutaneously in rats for 4 weeks. FIG. 11B shows the resultswith 25% collagen and 75% elastin implanted subcutaneously in rats for 4weeks.

Cross-linking has a number of benefits. (1) It decreases materialdegradation. (2) It decreases the swellability of elastin or other ECMparticles for better particle suspension and greater ease of injection(less clogging). (3) Proanthocyanidin is believed to increase fibroblastcellular activity; this should accelerate the regeneration process. (4)Proanthocyanidin is also an antioxidant that should quench free radicalsgenerated by local inflammatory reactions.

In the results presented herein, cross-linking with epoxide was found tobe more efficient in inhibiting material degradation. However,cross-linking to hyaluronic acid as described above was more effectivein inducing cellular activity of fibroblasts.

The results presented herein also show that, when elastin is used, ifthe elastin particle size is above 50 μm which is greater than the sizethat macrophages can engulf, elastin in general is stable subcutaneouslyor intracutaneously. When an elastin particle surface is cross-linkedwith a soluble layer of collagen or hyaluronic acid, there can beseveral effects. First, the cross-linking can change the morphology orcharge of the elastin surface, which can result in a better scaffold forregenerative cells such as fibroblasts to anchor, proliferate anddeposit ECM. Secondly, it is possible that a change in the surfacecharged produced by cross-linking can enable the particles to be bettersuspended in a pharmaceutically acceptable carrier.

If insoluble collagen is used as a particle phase, cross-linkingimproves the persistence of the injected or implanted composition. Ifthe collagen is from a xenogeneic source, cross-linking of the collagenalso reduces immunogenicity.

Advantages of the Invention

The present invention provides improved compositions and methods fordermal expansion and tissue filling that are effective in both cosmeticand reconstructive contexts. These compositions and methods areeffective, produce long-lasting results, and are well tolerated withoutmigration of the compositions or other significant side effects. Theinflammation that can occur is a physiological process that stimulatescollagen production, leading to an improved result and elimination ofwrinkles and other undesirable skin surface characteristics.

Compositions and methods according to the present invention possessindustrial applicability for the production of a medicament for use as adermal expander and tissue filler.

With respect to ranges of values, the invention encompasses eachintervening value between the upper and lower limits of the range to atleast a tenth of the lower limit's unit, unless the context clearlyindicates otherwise. Moreover, the invention encompasses any otherstated intervening values and ranges including either or both of theupper and lower limits of the range, unless specifically excluded fromthe stated range.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of ordinary skillin the art to which this invention belongs. One of ordinary skill in theart will also appreciate that any methods and materials similar orequivalent to those described herein can also be used to practice ortest this invention.

The publications and patents discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention. Further the dates of publication provided may bedifferent from the actual publication dates which may need to beindependently confirmed.

All the publications cited are incorporated herein by reference in theirentireties, including all published patents, patent applications, andliterature references, as well as those publications that have beenincorporated in those published documents. However, to the extent thatany publication incorporated herein by reference refers to informationto be published, applicants do not admit that any such informationpublished after the filing date of this application to be prior art.

As used in this specification and in the appended claims, the singularforms include the plural forms. For example the terms “a,” “an,” and“the” include plural references unless the content clearly dictatesotherwise. Additionally, the term “at least” preceding a series ofelements is to be understood as referring to every element in theseries. The inventions illustratively described herein can suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. As used herein, the transitionalphrase “comprising” is to include the transitional phrases “consistingessentially of” and “consisting of” unless the narrower transitionalphrases are expressly excluded in a particular context. Additionally,the terms and expressions employed herein have been used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of the futureshown and described or any portion thereof, and it is recognized thatvarious modifications are possible within the scope of the inventionclaimed. Thus, it should be understood that although the presentinvention has been specifically disclosed by preferred embodiments andoptional features, modification and variation of the inventions hereindisclosed can be resorted by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthe inventions disclosed herein. The inventions have been describedbroadly and generically herein. Each of the narrower species andsubgeneric groupings falling within the scope of the generic disclosurealso form part of these inventions. This includes the genericdescription of each invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised materials specifically resided therein. In addition, wherefeatures or aspects of an invention are described in terms of theMarkush group, those schooled in the art will recognize that theinvention is also thereby described in terms of any individual member orsubgroup of members of the Markush group. It is also to be understoodthat the above description is intended to be illustrative and notrestrictive. Many embodiments will be apparent to those of in the artupon reviewing the above description. The scope of the invention shouldtherefore, be determined not with reference to the above description,but should instead be determined with reference to the appended claims,along with the full scope of equivalents to which such claims areentitled. Those skilled in the art will recognize, or will be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described. Such equivalentsare intended to be encompassed by the following claims.

We claim:
 1. An injectable composition having dermal filling and tissueexpanding activity comprising: (a) a quantity of elastin sufficient tobring about dermal filling and tissue expansion when injected into asubject in need of dermal filling and tissue expansion; (b) apharmaceutically acceptable carrier, wherein the carrier comprises anexcipient selected from the group consisting of glucose, maltose,gelatin, and carboxymethylcellulose; (c) collagen; wherein thecomposition provides space-filling activity owing to the quantity ofelastin; wherein the quantity of elastin is from 20 mg/mL to 300 mg/mL;and wherein the elastin is full-length elastin.
 2. The composition ofclaim 1 wherein the collagen is in soluble form, fibrillar form, a formof an insoluble slurry, or hydrogel form.
 3. The composition of claim 1wherein the collagen is cross-linked intramolecularly.
 4. Thecomposition of claim 1 wherein the composition further comprises aglycosaminoglycan, wherein the glycosaminoglycan is selected from thegroup consisting of hyaluronic acid, chondroitin sulfate, pentosanpolysulfate, dermatan sulfates, heparin, heparan sulfates, and keratansulfates.
 5. The composition of claim 1 wherein the collagen iscrosslinked to the elastin or is crosslinked to a glycosaminoglycan,wherein the glycosaminoglycan is selected from the group consisting ofhyaluronic acid, chondroitin sulfate, pentosan polysulfate, dermatansulfates, heparin, heparan sulfates, and keratan sulfates.
 6. Thecomposition of claim 1 wherein the elastin is selected from the groupconsisting of non-human mammalian elastin and human elastin.
 7. Thecomposition of claim 1 wherein the pharmaceutically acceptable carriercomprises gelatin.
 8. The composition of claim 1 wherein the compositionfurther comprises a rapid-acting local anesthetic selected from thegroup consisting of lidocaine, benzocaine, tetracaine, bupivacaine,etidocaine, flecainide, mepivacaine, pramoxine, prilocaine,chloroprocaine, proparacaine, ropivacaine, dyclonine, dibucaine,propoxycaine, chloroxylenol, cinchocaine, dexivacaine, diamocaine,hexylcaine, levobupivacaine, pyrrocaine, risocaine, and rodocaine. 9.The composition of claim 8 wherein the rapid-acting local anesthetic islidocaine.
 10. The composition of claim 1 wherein the elastin iscrosslinked with a cross-linking agent selected from the groupconsisting of a compound that is a member of the class ofproanthocyanidins, a bifunctional epoxide, a carbodiimide, andglutaraldehyde.
 11. The composition of claim 10 wherein thecross-linking agent is a compound that is a member of the class ofproanthocyanidins.
 12. The composition of claim 11 wherein the compoundthat is a member of the class of proanthocyanidins is selected from thegroup consisting of proanthocyanidin, procyanidin(2H-1-benzopyran-3,4,5,7-tetrol,2-(3,4-dihydroxyphenyl)-2-((2-(3,4-dihydroxyphenyl)-3,4-dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl)oxy)-3,4-dihydro),procyanidin B, procyanidin B2, rhatannin, procyanidol oligomer,procyanidin C, procyanidin B3, procyanidin B1, selligueain A(8,14-methano-2H,14H-1-benzopyrano(7,8-d)(1,3)benzodioxocin-3,5,11,13,15-pentol,4-(3,4-dihydro-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-2H-1-benzopyran-8-yl)-3,4-dihydro-2,8-bis(4-hydroxyphenyl)-,(2R-(2α,3α, 4β(2R*, 3S*), 8β, 14β,15R*)), geranin A, geranin D, procyanidin B5,procyanidin B5-3′-O-gallate, vitisinol, amurensisin, terminalin, geraninB, 6,8-dihydroxyafzelin, afzelin-3″-O-gallate, geranin C, afzelin,flavangenol, carallidin, mahuannin A, proanthocyanidin A1,proanthocyanidin A2, and procyanidin D.
 13. The composition of claim 12wherein the compound that is a member of the class of proanthocyanidinsis proanthocyanidin.
 14. The composition of claim 10 wherein thecross-linking agent is a bifunctional epoxide and wherein thebifunctional epoxide is 1,4-butanediol glycidyl ether.
 15. Thecomposition of claim 10 wherein the cross-linking agent is acarbodiimide selected from the group consisting of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide (EAC);1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl-carbodiimidemetho-p-toluenesulfonate (CMC), andN-benzyl-N′-3-dimethylaminopropylcarbodiimide hydrochloride (BDC). 16.The composition of claim 10 wherein the cross-linking agent is selectedfrom the group consisting of glutaraldehyde and periodate.
 17. Thecomposition of claim 1 wherein the elastin is crosslinked with aglycosaminoglycan selected from the group consisting of hyaluronic acid,chondroitin sulfate, pentosan polysulfate, dermatan sulfates, heparin,heparan sulfates, and keratan sulfates.
 18. The composition of claim 1wherein the elastin is cryofractured into a fine particle.
 19. Thecomposition of claim 1 wherein the collagen is native or denaturedcollagen obtained from skin, tendon, ligament, pericardium, duralmembrane, small intestine mucosa, or bone.
 20. The composition of claim1 wherein the collagen is human Type I collagen.
 21. An injectablecomposition having dermal filling and tissue expanding activitycomprising: (a) an insoluble collagen suspension in a quantitysufficient to bring about dermal filling and tissue expansion wheninjected into a subject in need of dermal filling and tissue expansion;and (b) a pharmaceutically acceptable carrier.
 22. The composition ofclaim 21 wherein the concentration of collagen is from about 20 mg/mL toabout 100 mg/mL.
 23. The composition of claim 21 wherein the collagen iscross-linked with a cross-linking agent selected from the groupconsisting of a compound that is a member of the class ofproanthocyanidins, a bifunctional epoxide, a carbodiimide, andglutaraldehyde.
 24. The composition of claim 23 wherein thecross-linking agent is a compound that is a member of the class ofproanthocyanidins.
 25. The composition of claim 24 wherein the compoundthat is a member of the class of proanthocyanidins is selected from thegroup consisting of proanthocyanidin, procyanidin(2H-1-benzopyran-3,4,5,7-tetrol,2-(3,4-dihydroxyphenyl)-2-(2-(3,4-dihydroxyphenyl)-3,4-dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl)oxy)-3,4-dihydro),procyanidin B, procyanidin B2, rhatannin, procyanidol oligomer,procyanidin C, procyanidin B3, procyanidin B1, selligueain A(8,14-methano-2H,14H-1-benzopyrano(7,8-d)(1,3)benzodioxocin-3,5,11,13,15-pentol,4-(3,4-dihydro-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-2H-1-benzopyran-8-yl)-3,4-dihydro-2,8-bis(4-hydroxyphenyl)-,(2R-(2α,3α,4β(2R*,3S*),8β,14β,15R*)), geranin A, geranin D, procyanidin B5, procyanidinB5-3′-O-gallate, vitisinol, amurensisin, terminalin, geranin B,6,8-dihydroxyafzelin, afzelin-3″-O-gallate, geranin C, afzelin,flavangenol, carallidin, mahuannin A, proanthocyanidin A1,proanthocyanidin A2, and procyanidin D.
 26. The composition of claim 25wherein the compound that is a member of the class of proanthocyanidinsis proanthocyanidin.
 27. The composition of claim 23 wherein thecross-linking agent is a bifunctional epoxide and wherein thebifunctional epoxide is 1,4-butanediol glycidyl ether.
 28. Thecomposition of claim 23 wherein the cross-linking agent is acarbodiimide selected from the group consisting of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide (EAC);1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl-carbodiimidemetho-p-toluenesulfonate (CMC), andN-benzyl-N′-3-dimethylaminopropylcarbodiimide hydrochloride (BDC). 29.The composition of claim 23 wherein the cross-linking agent is selectedfrom the group consisting of glutaraldehyde and periodate.
 30. Thecomposition of claim 21 wherein the pharmaceutically acceptable carriercomprises an excipient selected from the group consisting of glucose,maltose, gelatin, and carboxymethylcellulose.